Through November 18-24, global and U.S. public health organizations (i.e., CDC and the WHO) focus their message on creating awareness about antimicrobial resistance. Based on CDC surveys, a staggering 2.8 million people suffer from antimicrobial-resistant infections in the U.S. every year.
Among antimicrobials, beta-lactams figure prominently in modern antibiotics used in the clinic. The discovery of penicillin in the 1920s by Sir Alexander Fleming and its introduction to the clinic in the 1940s represented an outstanding achievement for controlling bacterial infections. However, the quick rise of bacterial resistance to penicillin in the 1950s and subsequent beta-lactams, such as methicillin in the 60s, meant a new approach was critically needed (Ventola 2015). Vancomycin, a cyclic glycopeptide, was initially considered the answer to bacterial resistance. Yet, by the 80s, resistance to this “last-line” of defense antibiotic was also evident.
Beta-lactam antibiotics kill bacteria by targeting cell wall formation, specifically by binding and inhibiting bacterial enzymes necessary for peptidoglycan crosslinking. However, under the intense pressure of antibiotic misuse, bacteria have rapidly evolved many protective mechanisms, such as beta-lactamases, which hydrolyze the beta-lactam ring and inactivate these antibiotics (Bush and Bradford, 2016).
Mechanisms of bacterial resistance to beta-lactam antibiotics. Beta-lactamases target the beta-lactam ring for degradation in antibiotics, such as penicillin. Beta-lactamases are encoded by chromosomal and mobile genetic elements, and represent the major mechanism for Gram-negative bacterial resistance. Retrieved without modifications from (Vrancianu et al. 2020). https://creativecommons.org/licenses/by/4.0/
To tackle this resistance mechanism head-on, beta-lactamase inhibitors have been used with beta-lactam antibiotics as a combined therapy to boost the antibiotic’s efficacy. One key benefit of this strategy is the potential to reuse beta-lactam antibiotics that were otherwise made ineffective by bacterial resistance (Gonzalez-Bello et al. 2022). Nevertheless, among the hundreds of bacterial beta-lactamases identified to date, some fall within a group of extended-spectrum, which can target and inactivate beta-lactamase inhibitors, thus negating the benefits of combined therapies.
Generally, beta-lactamase inhibitors target the enzyme’s active site, out-competing other substrates and inactivating the enzyme through covalent binding. In recent work, a group at Switch Laboratory, Department of Cellular and Molecular Medicine, KU Leuven, Belgium, recognized that targeting the beta-lactamase active site might expedite more resistance to inhibitors and thus developed a new strategy to inhibit these enzymes (Khodaparats et al. 2023).
Khodaparats and colleagues have stirred away from the enzymes’ active site and instead have zeroed in on beta-lactamase aggregation-prone regions (APRs). Because APRs’ sequences are unique to specific proteins and conserved among beta-lactamase variants, Khodaparats et al. envisioned a strategy to specifically target them and promote their aggregation and, thereby, inactivation.
The team reasoned that APR-encoding synthetic peptides could be used to seed or induce beta-lactamase aggregation and misfolding, similar to the process of amyloid-like aggregation.
“We obtained these peptides from solid phase synthesis, followed by HPLC purification to a purity judged by reverse phase chromatography of at least 90% (Genscript).” Khodaparats et al. 2023.
To target extended-spectrum beta-lactamases, such as TEM-1 and SHV-11, Khodaparats et al. developed synthetic peptides following their previously established principles for the “Pept-In™” design. The Switch laboratory’s website states, “In their most basic design, Pept-Ins™ contain tandem repeats of an aggregation prone region flanked by gatekeeper residues and joined by a linker.” Based on this, Pept-Ins™, specifically targeting TEM-1 and SHV-11 variants, consisted of identified APR tandem repeats connected via a linker and a select number of residues to ensure solubility and bacterial peptide uptake.
Beta-Lactamase Pept-In Design Principles. The design of synthetic amyloid peptides by the Switch laboratory consisted of tandem APR sequence repeats of ~7 residues and followed their previous Pept-In™ design, incorporating arginine residues and a proline residue as a linker. Adapted from https://switchlab.org/technology/short-stretch-hypothesis-protein-aggregation/
TEM-1 and SHV-11 Pept-Ins™ demonstrated specificity, enhancing the effectiveness of beta-lactams (i.e., Penicillin G) only in E. coli resistant strains expressing the matching TEM or SVH beta-lactamases.
Interestingly, Pept-Ins™ designed to combine an APR from each, TEM-1 and SHV-11; beta-lactamase variant could augment the effectiveness of the antibiotic in both E. coli strains. Additionally, this combined peptide improved antibiotic performance against most TEM and SHV-expressing E. coli clinical isolates tested, demonstrating the versatility of this approach.
In vitro, the combined Pept-In™ (TEM/SHV) induced the expected protein aggregation effect and consequent reduction of beta-lactamase activity. In TEM-expressing E. coli cultures, peptide uptake was efficient and localized to inclusion bodies, indicative of protein aggregation that the team confirmed via Western Blot analysis.
Lastly, in vivo studies leveraging a mouse model of urinary tract infection with a TEM-1 beta-lactamase expressing E. coli strain demonstrated the feasibility of this strategy. Pept-In™ (TEM/SHV) was not toxic to mice following administration through different parenteral routes and improved the outcome of beta-lactam treatment, significantly reducing infection load and even outperforming the benefits of combined therapy with beta-lactam and the beta-lactamase inhibitor tazobactam.
Overall, the work of these investigators at the Switch Laboratory is opening the door to re-purposing a wide range of beta-lactam molecules previously abandoned at the clinic in the face of increasing bacterial resistance. These novel synthetic amyloidogenic peptides target conserved regions away from the rapidly evolving active site of beta-lactamases, which could help curb resistance issues. Finally, synthetic peptides represent a practical and accessible therapeutic modality with several advantages over small molecule inhibitors, such as better protein binding, higher specificity, lower tissue accumulation, and lower toxicity.
